Allergies are caused by an abnormality in the immune system that reacts by producing IgE antibodies to proteins which, per se, are completely harmless and mainly contained in pollens, acari, epithelia, and some foods.
Recent estimates show that more than 25% of people in industrialized nations are affected by this disease, that, persisting in time, may induce worsening of symptoms (e.g., onset of asthma), and sensitization to other allergens, thus making the choice of the most appropriate therapy more complicated (1).
Hyposensitizing specific immunotherapy (SIT), unlike pharmacological therapy, is the only form of etiological treatment of allergic diseases that is able to positively affect some immunological parameters that are the basis of the disease.
SIT consists of administration of increasing doses of standardized extracts (vaccines) obtained from the same substance causing the disease. In this way, a sort of “immunological tolerance” towards such substance is gradually induced in patients. This immunological tolerance is associated to a reduction or even disappearance of allergic symptoms.
The risk for eliciting side effects that are even severe in nature (2), also if it is considerably reduced by the use of slow-release vaccines or vaccines that are administered by alternative routes to injections, has limited SIT applicability in the therapy of allergic diseases. Moreover, as SIT is carried out by administering a mix of allergenic and non-allergenic proteins of natural origin without taking account of patient's sensitization profile, new IgE reactivities to initially harmful allergens that are present in the extract can arise.
The success of SIT is associated with a modulation of the immune response to allergenic molecules at the level of T helper cells and by the induction of blocking IgG antibodies specific for sensitising allergens. These (protective) antibodies may compete with IgE for antigen, influencing the tridimensional structure of this molecule, and inhibiting the IgE-mediated presentation of allergens to T cells, thus they interfere with cronic manifestation of atopy (3).
The development of vaccines made of recombinant proteins having less allergenicity but an unaltered immunogenic capacity might result in a further improvement in the field of allergic disease therapy.
During the last 15 years, considerable advances have been made in the field of allergen characterization thanks to the application of recombinant DNA-based technologies. cDNAs coding for main allergens are available for allergenicity studies and the development of diagnostic tests, and enable innovative SITs based on the use of purified recombinant proteins and their genetically modified variants that are characterized by reduced allergenic activity.
In the world, at least 40% of type 1 allergic patients are sensitised against pollen of grass genera belonging to the numerous family of Poaceae. About 20 species out of 9000, from five sub-families, are considered to be the most frequent cause of grass pollen allergy; the Poideae sub-family, in particular, is one of the major source of allergens in cool temperate and densely populated regions in North America, Europe and southern parts of Australia because it is largely distributed and able to produce large amounts of pollen (4).
The grass pollen allergens have been classified in different groups according to their cross-reactivity. Up to 13 families of allergens have been described within Pooideae, out of which group 1 and 5 allergens appear to be the most clinically relevant, since 95 and up to 85% of patients with grass allergy, respectively, are sensitized to them. IgE antibodies against these two classes of major allergens represent 80% or more of specific IgE in patients' sera.
Group 1 allergens are glycoproteins with a molecular weight around 30-35 kDa, with similar biochemical and functional properties to β-expansins, enzymes belonging to the family of cystein-proteinases involved in the processes of growing, differentiation, fertilization and ripening of the fruits. Sequencing of the group 1 allergens from eight species has revealed a 90% level of amino acid conservation.
Group 5 allergens are non-glycosylated proteins with a molecular weight around 28-32 kDa which share a high degree of amino acid sequence identity (55-80%) which confers them cross-reactivity in both IgE binding and T-cell reactive epitope; one of their members (Phl p Vb) was reported to have ribonuclease activity.
Within Pooideae sub-family, Phleum pratense pollen is the most studied, because it represents an important cause of rhinitis, conjunctivitis and bronchial asthma (5); this pollen has been often utilized as source for allergen isolation and cloning, and can be considered representative of the whole sub-family and suitable for allergen-specific immunotherapy against allergy to Poideae grass pollens (6).
The two main allergens in Phleum pratense pollen are Phl p 1 (whose nucleotide sequence is identified by GenBank Acc. No. X78813) and Phl p 5 (7).
Phl p 5 is represented by several isoforms which can be discerned in two main groups denoted a (AF069470) (8) and b (Z27083). Incubation of natural Phl p 5, separated by 2D electrophoresis, with specific IgE antibodies revealed that each one of the two isoforms is divided in at least four isoallergens bearing identical epitopes but at least one different epitope, located on both the C-terminal and N-terminal of the molecule.
In recent years, most attention has been focused on the development of safer, more effective vaccines, consisting of recombinant proteins mutagenised at the level of amino acids important for IgE binding, namely hypoallergenic variants capable of favourably influencing the natural progression of the disease without causing undesired side effects (9).
Numerous studies have been performed to identify or alter IgE epitopes of major allergen Phl p 5 from Phleum pratense or of omologous proteins produced in other grasses belonging to the Pooideae sub-family.
Concerning Phl p 5b isoform, conformational nature of some IgE epitopes was shown. Using site-directed mutagenesis, 10 surface-exposed lysines out of the 12 lysines located in the C-terminal half of the molecule were substituted by alanines. IgE binding capacity of lysine-deficient mutant was significantly diminished as demonstrated by ELISA inhibition tests and basophil stimulation, and in vivo skin prick tests (SPT). The ability to induce specific IgG antibodies was unchanged demonstrating that substitution of those lysine amino acids did not hamper structural protein integrity (10).
The construction of point and deletion Phl p 5b mutants showed that IgE-binding capacity of the molecule was reduced in dependence of the length of the deletion. Mutants lacking parts of the molecule, like the stretches of aminoacids 49-133 (DM1), 50-178 (DM2), 153-178 (DM3), 71-91 coupled with 153-178 (DM4), or carrying the following point mutations A13C, N32D, N38D, D49L, K50A, A156T, A220T, A241T were analysed. Introduction of these point mutations or the shorter deletion DM3 did not affect IgE-binding capacity of these variants, in contrast the larger deletions in DM2 and DM4 mutants caused a strong reduction of IgE reactivity in vitro (by ELISA inhibition and histamine release assay) and in vivo (SPT). Mutants lacking individual epitopes were not able to induce proliferation of T cells specific for these epitopes. The best compromise was found in the case of mutant DM4, where autors observed a nearly complete reduction of IgE-binding capacity together with an overall conserved cells reactivity (11).
Based on the successful results obtained by Schramm (11) and the high sequence homology displayed by the two isoforms, the effect of some deletions studied in Phl p 5b was tested on corresponding regions in Phl p 5a molecule. It was observed that double-deletion mutant Phl p 5a Δ(94-113, 175-198) showed strongly diminished IgE-binding in immunoblot assay and IgE-inhibition tests, and an 11.5-fold reduced capability to activate basophils compared with the recombinant wild-type molecule. T cell proliferation assays demonstrated retention of stimulating capacity of double-deletion mutant. The mutant characterised by single deletion Δ94-113, corresponding to elix 3 and located in the N-terminal of the molecule, showed a reduced IgE-binding only in a subgroup of allergic patients′sera and a reduced capacity to induce histamine release by basophils. Deletion Δ175-198 alone located in the C-terminal caused a higher reduction in IgE-binding than Δ94-113 deletion, but lower than double-deletion (12).
To confirm the presence of important epitopes in the N-terminal region of Phl p 5a, the fragment Phl p 5a (56-165), comprising about one-third of the full-length molecule, was characterised. This study demonstrated this fragment represents an immunodominant portion of the allergen and contains IgE epitopes, as it strongly reacts with serum IgE from 90% of tested grass pollen allergic patients (13).
Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, the sequence of Lol p 5, the group 5 allergen from ryegrass, was modified by site-directed mutagenesis in five highly conserved domains (D1-D5). Replacement of the aminoacids K57A (D1), K172N, F173L, T174A, V175A (D2), A204G, V205A, K206A (D3), G273A (D4), K275A (D5), combined in different ways to obtain multiple-mutants, led to a reduction of IgE binding capacity and allergenic activity, as determined by basophil histamine release and SPT in allergic patients, but did not affect the ability to induce T cell proliferation. This work demonstrated that an immunodominant B-cell epitope in Lol p 5 involves D2 domain (14).
The possibility to use engineered multimeric molecules consisting of different allergens from the same organism or different organisms for specific hyposensitising therapy has long been considered fascinating. This approach allows a number of allergens to be assembled in a single molecule with the advantage of producing a single preparation containing the allergens in a precise molar ratio.
It has been demonstrated that immunogenity of two hypoallergenic molecules can be increase by assembling them as a single hybrid molecule. Concerning grass allergy, the association of Phl p 1 and Phl p 5 in a single hybrid molecule should represent the T-epitope repertoire of both molecules, and may induce a strong IgG protective response against both allergens.
Linhart et al. (15) developed an engineered hypoallergenic molecule composed of Phl p 2 and Phl p 6 allergens, where Phl p 2 nucleotide sequence was disrupted by fragmentation, reassembled in altered order and fused with a truncated Phl p 6 sequence. The hybrid retained the reduction of IgE reactivity and allergenic activity of its components as shown by ELISA and basophil activation assays. Immunization of mice with the hybrid molecule demonstrated the increased immunogenicity of this hybrid molecule, leading to higher levels of allergen-specific IgG antibodies compared to the single components.
An engineered molecule was developed by assembling of cDNAs coding for the four major Phleum pratense allergens Phl p 1, Phl p 2, Phl p 5, Phl p 6. The hybrid contained most of the B-cell epitopes of grass pollen and could be used to diagnose allergy in 98% (n=652) of patients allergic to these plants. Immunization of mice and rabbits with the hybrid molecule induced stronger and earlier IgG antibody response than equimolar mixtures of the single components (16). Results from a later work confirmed the capacity of this hybrid, consisting of four major allergens, of replacing traditional Phleum pratense pollen extract for the in vivo diagnosis of grass pollen allergy by means of SPT (17).
As conventional immunotherapy is more effective in patients with allergic mono-sensitivities compared with multi-sensitised subjects, an allergen chimera was genetically engineered for treatment of multi-sensitisation to birch and grass pollen. The major birch pollen allergen Bet v 1 served as a scaffold for N- and C- terminal linkage of the immunodominant peptides of the major Phleum pratense pollen allergens Phl p 1 and Phl p 5. The chimera was immunogenic in mice for T and B cell responses to the three allergens. Intranasal application of the chimera prior to poly-sensitization significantly suppressed humoral and cellular Th2 responses specific for the three allergens and prevented development of airways inflammation upon allergen challenge with Phleum pratense and birch pollen extracts (18).
The data available in the literature show the regions where IgE epitopes of the grass pollen allergens, in particular belonging to the group 5, are located, without indicating the single aminoacids involved in IgE binding.
On the basis of current knowledge, most of the grass allergic patients is mainly sensitized against Group 1 and 5 allergens. The use of a highly purified hypoallergenic hybrid variant like Phl p 1-Phl p 5 might induce a targeted response to the two main allergens and eliminate the traditional therapy-related side effects caused by strong allergenic activity of crude extract and presence of numerous further molecules.
Contrary to the results proposed by Wild (18) where chimeric allergen exclusively included the immunodominant epitopes of Phl p 1 and Phl p 5, the association of the two complete allergens would represent the whole repertoire of T epitopes, and might induce a strong protective IgG response in a wider allergic population.